A number of heterologous expression systems have been devised over the last decade for the production of clinically and agronomically useful recombinant proteins. A significant challenge in most systems is to optimize the yield and quality of the recombinant protein product. Significant progress has been achieved over the last 15 years in the optimization of transgene transcription and translation in plants (Potenza et al., 2004, In Vitro Cell. Dev. Biol.-Plant, 40, 1-22; Streatfield, 2007, Plant Biotechnol. J. 5, 2-15) and the elucidation and modulation of the complex protein post-translational modifications characteristic of the plant cell machinery (Gomord and Faye, 2004, Curr. Opin. Plant Biol. 7, 171-181; Faye et al., 2005, Vaccine 23, 1770-1778). Despite these advances, ensuring satisfactory yield and quality of recombinant proteins often remains a difficult task.
One factor strongly influencing recombinant protein quality and yield is the relative inherent stability of polypeptide chains expressed in a heterologous environment (Faye et al., 2005).
Proteolytic enzymes, or proteases, contribute to the overall control of metabolic and transduction pathways by directing the activation or hydrolysis of proteins implicated in key regulatory processes, or by contributing to the elimination of misfolded proteins and the selective recycling of amino acids from short-lived proteins (Vierstra, 2003, Trends Plant Sci. 8, 135-142; Schaller, 2004, Planta, 220, 183-197). In plants, these enzymes also initiate the general recycling of proteins in senescing organs and the mobilization of amino acid constituents of seed or tuber storage proteins during germination (Müntz, 2007, J. Exp. Bot. 58, 2391-2407).
Proteases may affect the integrity of recombinant proteins in different ways, both in planta during protein expression and ex planta during extraction and subsequent downstream processing (Michaud et al., 1998, Methods Biotechnol. 3, 177-188; Rivard et al., 2006, Plant Biotechnol. J. 4, 359-368). Depending on the number of “susceptible” cleavage sites accessible to endogenous proteases for peptide bond hydrolysis, the protein may undergo complete hydrolysis directly impacting on its final yield or partial trimming, altering the activity or homogeneity of the final protein product. Although interesting yields may be obtained in terms of net protein levels, the final product may show altered integrity, structural heterogeneity and/or deficient biological activity, potentially altering its value for commercialization (Faye et al., 2005).